Mass spectrometry has been used for the identification of microorganisms (microbes) in cell cultures for many years.
Microbes can be grown on the surface of a solid medium, for example formed by combining liquid agar with appropriate nutrients and the mixture is then allowed to solidify on a sample plate. Samples of microbes are then deposited on the surface of the solid medium and the plate is stored for a period of time in controlled conditions favourable for the growth of the microbes.
The growth conditions for growing a particular sample of microbes, including for example the type and mixture of nutrients provided in the growth medium, the conditions such as temperature in which the microbes are incubated, and the length of time for which the microbes are incubated, can be selected based on one or more factors (e.g. on the type of microbes that are suspected to be present in the sample). One or more additional substances may be added to the growth medium to, for example, suppress the growth of certain microbes or classes of microbe in the sample or to enable easier distinction between the microbe sample from other microorganisms.
The identification of a microbe sample using mass spectrometry usually proceeds by transferring some of the microbe culture from the sample plate onto a mass spectrometry sample plate. A sample mass spectrum of the sample microbe can be obtained by Matrix Assisted Laser Desorption Ionisation (“MALDI”) Time of Flight (“ToF”) mass analysis. A matrix substance is added to the sample plate which is used in a subsequent ionization by a Matrix Assisted Laser Desorption Ionisation ion source to generate positively charged ions. The generated ions are then mass analysed using a Time of Flight mass analyser. Other types of ionization may be used including ion sources that generate negatively charged ions.
Multiple reference spectra (e.g. 10 or more) corresponding to a known type of microbe are usually acquired and stored. This is performed for a number of different types of microbes to create a library of reference spectra for a plurality of different known types of microbes. The multiple reference spectra of a particular type of microbe are intended to capture variability that arises when acquiring the reference spectra and/or in the experimental parameters.
Such variability arises for a number of reasons. For example, mass spectra acquired for a particular type of microbe may show differences due to factors that cannot be controlled e.g. systematic factors such as chemical, statistical or electrical noise. In addition, a particular microbe may produce significantly different reference mass spectra as a result of variations in conditions that can be controlled e.g. the conditions under which the microbe is grown, differences in the composition of the growth medium on which it is cultured, the length of time for which it is cultured and the temperature at which the culture is stored, etc. amongst many others factors. The individual mass spectrometer used to obtain the mass spectra of the microbe can also contribute to the variability.
It is desirable to incorporate as much of the variability as possible in the library by obtaining and storing as many reference spectra of a particular type of microbe as possible. However, if an excessive number of reference spectra corresponding to a variety of controllable conditions are obtained and incorporated in the library as a single “consolidated” reference spectrum, then mass peaks that are potentially useful for diagnostic purposes may be averaged out or eliminated.
In operation, a sample containing unknown microbes is analysed. Multiple mass spectra (replicates) of the sample are usually obtained and the obtained mass spectra are compared with the reference mass spectra in the library. In one class of identification strategy, a score or an index is assigned to every library entry (reference spectrum) which characterises the agreement or similarity between a reference spectrum and the mass spectrum obtained from the sample and reflects the goodness of fit of the reference spectrum with respect to the sample spectrum. The assigned scores or indices are then used to rank the library entries.
Amongst the reference spectra of a particular type of microbe, it is often arranged that components of the spectra that exhibit less variability contribute more to the score than those that are more variable. These less variable components are deemed to be more informative. Here, a “component” refers to a mass spectrometric peak, a mass region or an individual mass channel. Such selection may be carried out in a soft approach, for example through the use of likelihood of agreement, or in a more hard-edged approach, for example using the variability to select (or discard) peaks to be stored in the library.
US 2008/0300826 (Schweitzer) describes a method of searching a spectral database comprising plural sublibraries in order to identify unknown materials. A search of a sublibrary is performed using a similarity metric that compares a test data set to each reference data set of the sublibrary. The result is a set of probabilities for the sublibrary, with one probability for each reference data set of the sublibrary. This searching process is performed in respect of plural sublibraries, which each relate to a different analytical technique (e.g. Raman spectroscopy, fluorescence spectroscopy, mass spectrometry, etc.), using different sample spectra and reference spectra produced using the different analytical techniques. Corresponding probabilities for a particular reference material that are obtained using the different analytical techniques are then “fused” to give a set of final probabilities. The unknown sample is then classified based on the highest final probability.
GB 2471746 (Maier) describes a method of identifying microbes by comparison of their mass spectra with reference spectra. The method comprises calculating similarity indicators between the reference spectra and a sample spectrum.
US 2011/0202282 (Kostrzewa) discloses a multi stage search algorithm for identifying a sample of microbes wherein a library of reference spectra is divided into a number of sub-libraries, each sub-library being organised according to the frequency of identification of different species of microbes. The structure is used to improve searching speed by first searching through a sub-library of the most frequently identified species of microbes and then through sub-libraries of decreasing frequency.
It is desired to provide an improved method of analysing a sample mass spectrum.